Mitochondrial metabolite compartmentalization in health and disease

健康和疾病中的线粒体代谢物区室化

• 批准号: 10226352
• 负责人: Shingo Kajimura
• 金额: $ 87.5万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10226352
• 关键词: Adipocytes; Biological; Biological Process; Brown Fat; CRISPR interference; Carrier Proteins; Cells; Cues; Defect; Diabetes Mellitus; Disease; Escherichia coli; Eukaryotic Cell; Goals; Health; Heart; Human; Inner mitochondrial membrane; Insulin Resistance; Knowledge; Liposomes; Mammals; Maps; Mediating; Metabolic; Metabolic Diseases; Mitochondria; Mitochondrial Matrix; Mitochondrial Membrane Protein; Molecular; Mus; Nature; Obesity; Organelles; Orphan; Pathologic; Physiological; Process; Protein Family; Recombinant Proteins; Regulation; Role; Stress; System; Work; Yeasts; disease phenotype; enzyme substrate; flexibility; in vivo; metabolomics; reconstitution; stem

Project Summary Eukaryotic cells store and utilize metabolites in different organelles – referred to as subcellular metabolite compartmentalization. Distinct pools of metabolic enzymes and substrates provide another layer of flexibility in metabolite utilization, thereby allowing for robust adaptation to a variety of intrinsic cues and external stress. In turn, defects in the processes are associated with metabolic disorders, including obesity, insulin resistance, and diabetes. One of the critical regulators of metabolite compartmentalization is mitochondrial transporters: a large number of carrier proteins, many of which belong to the SLC25A protein family, mediate the translocation of metabolites across the impermeable mitochondrial inner-membrane and control their availability in the mitochondrial matrix. However, a vast majority of the mitochondrial SLC25A carrier proteins are “orphan” transporters, i.e., their specific substrates and biological functions remain unknown. The lack of our knowledge is primarily due to the fact that many mitochondrial membrane proteins cannot be reconstituted correctly in the conventional experimental system, i.e., liposomes using recombinant proteins made in E. Coli or yeast. To circumvent this issue, we developed a robust experimental platform that enables systemic characterization of mammalian mitochondrial transporters using brown fat, one of the most mitochondria- enriched cells. We incorporated the CRISPRi and CRISPRa system in immortalized brown adipocytes, such that we can obtain essentially unlimited amounts of “designer mitochondria” in mice and humans. By employing the new system, my lab has recently identified SLC25A44 as the first mitochondrial BCAA transporter in mammals, a long-standing mystery in the field (Yoneshiro et al. Nature 2019). This proposal aims to generate a complete functional map of mitochondrial SLC25A metabolite transporters in mammals. To achieve this goal, we plan to apply the state-of-art metabolomics and mitochondrial-liposomes to the brown fat-derived designer mitochondria, and to determine the specific substrates for orphan SLC25A carrier proteins. We will further determine the physiological and pathological roles of orphan SLC25A transporters in vivo, with an emphasis on metabolic disorders. The work resulting from this application will establish a conceptual framework to understand the molecular regulation of mitochondrial metabolite compartmentalization, and also provide a new roadmap for reversing disease phenotypes that stem from defects in such processes.

项目总结